Anti-human galactosyltransferase (E.C. 2.4.1.22) antibodies were elicited in rabbits and purified on a galactosyltransferase-agarose column. Purified antibodies were used to localize galactosyltransferase in acetone-fixed HeLa cells and human lung fibroblasts. Both protein A-peroxidase developed with 3-amino 9-ethylcarbazole and swine anti-rabbit lgG-fluorescein isothiocyanate served to detect binding of anti-galactosyltransferase antibodies. In cells of confluent cultures, anti-galactosyltransferase staining appeared as a concise triangular structure in the juxtanuclear region with one angle oriented

No abstract

Rabbit antisera against soluble human milk galactosyltransférase (GT) having anti-GT activity, as demonstrated by inhibition of enzyme activity were used for a comparative study of the molecular sizes of galactosyltransférase. For this purpose, affinity-purified antibodies were used for the identification of milk, serum and effusion galactosyltransférase from native or partially purified preparations resolved by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) by the immune replica technique. Milk galactosyltransférase migrated as a 55-kilodalton (kD) protein, serum and effusion GT slightly faster. Crossreactive enzyme forms of 110 kD and 20 kD were detected in milk only. In order to establish a relationship between intracellular and soluble galactosyltransférase, HeLa cells were metabolically labeled by [35S]-methionine, cells lysed, subjected to immunoprécipitation and the precipitate analyzed by SDS-PAGE/ftuorography: a single band corresponding to the intracellular form of GT having similar mobility as the milk enzyme was detected. These results indicate a close structural similarity between soluble and cellular galactosyltransférase as judged by immunological cross-reactivity and electrophoretic mobility.